A. Petiot et al., Distinct classes of phosphatidylinositol 3 '-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells, J BIOL CHEM, 275(2), 2000, pp. 992-998
3-Methyladenine which stops macroautophagy at the sequestration step in mam
malian cells also inhibits the phosphoinositide 3-kinase (PI3K) activity ra
ising the possibility that PI3K signaling controls the macroautophagic path
way (Blommaart, E. F. C., Krause, U., Schellens, J. P. M., Vreeling-Sindela
rova, H., and Meijer, A. J. (1997) fur. J, Biochem. 243, 240-246). The aim
of this study was to identify PI3Ks involved in the control of macroautopha
gic sequestration in human colon cancer HT-29 cells. An increase of class I
PISK products (phosphatidylinositol 3,4-bisphosphate and phosphatidylinosi
tol 3,4,5-triphosphate) caused by either feeding cells with synthetic lipid
s (dipalmitoyl phosphatidylinositol 3,4-bisphosphate and dipalmitoyl phosph
atidylinositol 3,4,5-triphosphate) or by stimulating the enzymatic activity
by interleukin-13 reduced macroautophagy, In contrast, an increase in the
class III PISK product (phosphatidylinositol 3-phosphate), either by feedin
g cells with a synthetic lipid or by overexpressing the p150 adaptor, stimu
lates macroautophagy. Transfection of a specific class III PISK antisense o
ligonucleotide greatly inhibited the rate of macroautophagy. In accordance
with a role of class III PISK, wortmannin tan inhibitor of PI3Ks) inhibits
macroautophagic sequestration and protein degradation in the low nanomolar
range (IC50 5-15 nM), Further in vitro enzymatic assay showed that 3-methyl
adenine inhibits the class III PISK activity. Dipalmitoyl phosphatidylinosi
tol 3-phosphate supplementation or p150 overexpression rescued the macroaut
ophagic pathway in HT-29 cells overexpressing a GTPase-deficient mutant of
the G alpha(i3) protein suggesting that both class III PISK and trimeric G(
i3) protein signaling are required in the control macroautophagy in HT-29 c
ells. In conclusion, our results demonstrate that distinct classes of PI3K
control the macroautophagic pathway in opposite directions. The roles of PI
3Ks in macroautophagy are discussed in the context of membrane recycling.